The industrial use of enzymes is often limited by their high cost and rapid inactivation. Soluble enzymes are lost with the product at the conclusion of a process, and must be replenished.
One means to improve the economic feasibility of enzymes for industrial processes is through enzyme immobilization onto a matrix, which may facilitate their re-use. Immobilization research has focused upon means to enhance the transfer of enzymes onto the support, and upon means to ensure that the immobilized enzymes remain active. Inactivation of enzymes during catalytic turnover is another key obstacle which limits the economic feasibility of enzyme-mediated processes. Enzymes may be inactivated by extremes of temperature, pH, shear, and also by free radicals and other reactive species present in the reaction medium. Immobilization technology has the potential to reduce such enzyme inactivation, and thus extend their useful lifespans.
Biochemical Engineering Journal, 4, (2000), 137-141, Ganesh K et al, teaches that during the production or downstream processing of an enzyme it is always subjected to shear stress, which may deactivate the enzyme. This susceptibility of enzymes to shear stress is a major concern as it leads to the loss of enzyme activity and is, therefore, a major consideration in the design of the processes involving enzyme production and its application. In this reference, cellulase enzyme was subjected to shear stress in a stirred reactor with an objective of investigating its deactivation under various conditions, such as different agitation speeds, concentrations of enzyme, concentrations of buffer, pH ranges, buffer systems and the presence of gas-liquid interface. It was found that the extent of deactivation depends upon the conditions under which the enzyme was subjected to shear.
For industrial use, it is generally not sufficient for only one of these obstacles to be overcome. For example, a stable, immobilized enzyme that cannot be easily recovered or reused offers few advantages. Similarly, an immobilized enzyme that is easily recovered and reused, but does not maintain its activity over an extended period offers few advantages over a process mediated by soluble enzymes, since, in both cases, the enzymes must be replenished at frequent intervals. The main goals are to produce a stable immobilized enzyme, which can also be efficiently and completely recovered, so that its useful lifespan is many times greater than the single use afforded by a soluble enzyme. An enzyme recovery system is therefore of paramount importance.
To date, immobilized enzyme/reactor technologies have focused on “in situ” enzyme preparations, in which the enzymes are retained within the reactors, while the process fluid is passed through. This prevents loss of the enzyme with the process fluid, and allows the enzyme to be used several times. Examples include, enzymes immobilized within gels which are used within a packed bed reactor, and enzymes attached to, or retained within, semi-permeable, hollow, fiber membranes. Enzymes have also been incorporated within monoliths, such as those used in an automobile catalytic converter, wherein the fluid containing the substrate passes through the monolith.
Unfortunately, enzymes “entrapped” within gels are subject to mass transfer limitations, which may dramatically reduce the performance of the immobilized enzymes, relative to soluble enzymes. The use of enzymes in packed beds is seemingly attractive in that the immobilized enzyme is retained within the reactor, while the process fluid containing substrate and product passes through. However, such an arrangement may be limited by mass transfer outside the particle, due to restriction of fluid flow around the tightly packed particles. To avoid extra-particle mass transfer limitations, relatively large particles are used for the immobilized enzyme. However, larger particles lead to greater mass transfer limitations within the particle, and consequently, it is necessary to choose a particle size that balances intraparticle and extraparticle mass transport. Furthermore, a packed-bed arrangement is only practical if the substrate is easily transported through the packed bed. Replacing the immobilized enzyme in such a reactor may also be time-consuming as to lead to significant process “down-time”. Thus, the economic benefits are only realized if the enzyme is very stable, and it does not need frequent replacement.
Packed bed reactors and other types of “in situ” immobilized enzyme reactors, such as monoliths and hollow fibers, can be prone to plugging. Consequently, they may be inappropriate if the substrate is insoluble, for e.g., in a slurry. In all cases, in situ preparations rely on the transport of the substrate to the immobilized enzyme, either by convection or diffusion. If flow is “segregated”, as in a slurry, or there is insufficient mixing, or if the substrates are bulky and have low diffusion rates, such substrate-enzyme contact may be hindered and lead to dramatically reduced efficiency and performance. Since some of the key industrial enzymatic processes involve slurries, e.g., starch hydrolysis and pulp processing, there is a need for an immobilized enzyme reactor process that differs from the traditional “in situ” immobilized enzyme reactor.
A particular example of an immobilized enzyme reactor is that which is used for isomerization of dextrose to fructose. A solution of soluble dextrose passes through a bed of immobilized glucose isomerase at such a rate as to ensure a specific product, namely, fructose, concentration at the bed outlet. Owing to continuous inactivation of the immobilized enzyme, the flow rate through the packed bed reactor must be continuously reduced, to ensure that the fructose concentration of the effluent is held constant. It is often necessary to reduce the feed flow rate by a factor of ten or more throughout the “useful” lifespan of the immobilized enzyme. However, such a reduction in flow rate also leads to a proportional reduction in the rate of production of fructose. Consequently, an array of generally 20 or more reactors is used, each of which reactor contains immobilized glucose isomerase of a different age, and each with a different, continuously changing flow rate (H. S. Olsen, Enzymatic Production of Glucose Syrups, in Handbook of Starch Hydrolysis Products and Their Derivatives, M. W. Kearsley and S. Z. Dziedzic, eds., Blackie Academic and Prof. Publishers (Chapman and Hall), Glasgow, 1995). By combining the effluent from each reactor, the average production rate of fructose is kept relatively constant. Once the immobilized enzyme reaches the end of its useful lifespan, that reactor in the array is taken out of service, and the immobilized enzyme is replaced with fresh enzyme.
Unfortunately, such a process arrangement is cumbersome and complex, and the capital cost is high due to the number of reactors required, and the need for complex valving and process control equipment to manage the adjustment of fluid flow rates to each reactor in the array. A further disadvantage of such an arrangement is the production of “color” and other “off-flavor” reversion byproducts, which are more likely to be generated at low flow rates and thus, high residence times. Thus, improvements in immobilized enzyme technology and enzyme recovery methods could dramatically simplify this process, and improve its economics.
U.S. Pat. No. 5,177,005, issued Jan. 5, 1993—Lloyd and Antrim, describes a continuous process for production of fructose involving glucose isomerase adsorbed onto a support such as a resin. The reactor was packed with excess resin as a support and, periodically, fresh, soluble glucose isomerase was added to compensate for the inevitable loss of activity of the previously immobilized enzyme. In trials, fresh glucose isomerase was added approximately every 3 weeks, on average, to keep the dextrose conversion between 40 and 44%. Over a 27 week trial, the quantity of soluble enzyme added represented approximately 4 times the quantity of enzyme originally present in the reactor. Unfortunately, such additions can only continue as long as there is binding capacity on the resin. Once the resin is fully loaded, additional soluble glucose isomerase may confer little benefit, unless the inactivated enzyme is somehow removed from the support. At this point, the process must either be shut down to replace the gel, or run in a “variable flow rate mode”, similar to that with traditional immobilized glucose isomerase, described hereinbefore.
U.S. Pat. No. 4,033,820, issued Jul. 5, 1977—Brouillard R. E. describes another in situ immobilized enzyme preparation based on a highly porous, spongy starch gel, modified to act as a support for the enzymes. It was acknowledged that this approach is only suitable for substrates that are soluble in water and that slurries cannot be processed. Another challenge with this approach is the degradation of the starch support gel. Several exotic cross-linking treatments are required if the support is to be used for enzymes that degrade starch, e.g., amylase and glucoamylase. Bactericides, such as formaldehyde or chlorine, were used to regularly wash the column to prevent bacterial contamination.
U.S. Pat. No. 4,209,591, issued Jun. 24, 1980 to Hendriks P., describes a multistage fluidized bed process involving countercurrent flow of an immobilized enzyme and substrate solution. The system is designed such that nearly all of the activity of the immobilized enzyme has been lost by the time it reaches the outlet of the multistage unit. Thus, this operation also involves a “single use” of enzyme, since the enzyme is not recovered or reused. Sieve plates or mesh screens may be used between and within stages to regulate the transfer of immobilized enzyme and substrate from one compartment to the next. In principal, this process is amenable to the use of substrate slurries, however, the particle sizes of the substrate and immobilized enzyme must be small enough to pass through the sieve or mesh. Furthermore, an extremely narrow particle size distribution is required for the immobilized enzyme, to prevent its separation into various sized fractions during operation. It is well accepted that for proper fluidization, the flow rate of the substrate solution is determined in large part by the size and shape of particles and the particle density relative to that of the fluid.
Other examples aimed at multiple use of immobilized enzymes include U.S. Pat. No. 4,442,216, issued Apr. 10, 1984 to Harvey, D. G., U.S. Pat. No. 4,511,654, issued Apr. 16, 1985 to Rohrbach et al, and U.S. Pat. No. 4,594,322, issued Jun. 10, 1986 to Thompson G. J. The former describes a screw-type reactor that includes a screw-lift mechanism and conveyor to recover the immobilized enzyme, wash it and return it to the inlet of the reactor. U.S. Pat. No. 4,511,654 describes an immobilized glucoamylase packed bed reactor with subsequent substrate recycle via an ultrafiltration membrane. The enzyme in this process is, thus, in situ. This process is suitable for soluble sugar feedstocks only. Aforesaid U.S. Pat. No. 4,594,322 describes a similar process in which the hydrolysis products are separated into a glucose-rich stream and a polysaccharide-rich stream, the latter of which is recycled to pass again through the reactor containing immobilized enzyme.
U.S. Pat. No. 4,844,809, issued Jul. 4, 1989 to Yoshiro et al, describes the use of a hollow fiber membrane for removal of fine particles from a reaction solution. While the inventors cite this invention for other purposes, such as the removal of impurities, such a technique could also, conceivably, be used to retain enzymes immobilized to fine particles within a hollow fiber reactor, e.g. “Ultrafiltration Separation of Cellulase and Glucose for a Lignocellulosic Biomass to Ethanol Process”, J. S. Knutsen and R. H. Davis, Conference Proceedings, Symposium on Biotechnology for Fuels and Chemicals, Breckenridge, Colo., May. 6-9, 2001.
Consequently, applications of immobilized enzymes have been essentially restricted to in situ preparations, usually within packed bed reactors. Such processes, although technically and, occasionally economically feasible, may be unnecessarily cumbersome. They may also be unsuitable for process streams in which the substrate is present as a slurry. An immobilized enzyme process capable of processing slurries, and/or which avoids the complexity required to account for enzyme inactivation within in situ preparations, e.g. production of HFCS, would be extremely advantageous.
Slurries cannot be processed using an in situ immobilized enzyme within a packed bed or monolith reactor, due to plugging of the bed or monolith, and mass transfer problems that limit immobilized enzyme-substrate contact. A design in which the immobilized enzyme is free to circulate within the reactor can overcome these limitations. However, a means to facilitate recovery and reuse of the immobilized enzyme is also required.
There is, therefore, a need for an immobilized enzyme recovery process which enables the immobilized enzyme to be reused and, preferably, recycled within a full enzymatic treatment plant, wherein substrate feedstock is enzymatically treated with particulate immobilized enzyme.